In insects, amine acetylation by the enzyme arylalkylamine N-acetyltransferase (AANAT) is involved in melatonin formation, sclerotization, and neurotransmitter inactivation. This wide spectrum of activities suggests that several AANAT enzymes are present. We recently purified a protein fraction with AANAT activity from Drosophila melanogaster and cloned the corresponding gene, aaNAT1. Following the same strategy, we now report the purification of an additional AANAT from D. melanogaster, AANAT2, and the cloning of the corresponding cDNA. The isolated protein differs from AANAT1a and AANAT1b in its molecular weight and isoelectric point. The AANAT2 shares about 30% identity with the products of the aaNAT1 gene. The enzyme does not follow one-site Michaelis-Menten kinetics when assayed with various concentrations of the arylalkylamine tryptamine and a constant concentration (0.5 mM) of the cofactor acetyl coenzyme A. The data can be interpreted in terms of an enzyme with two kinetic regimes (K(m1) = 7.2 microM, K(m2) = 0.6 mM, and v(max2) = 2.7 v(max1)) that are governed by binding of the substrate to a regulatory site (K(r) = 6.2 mM). These findings demonstrate the presence of a second expressed gene encoding an AANAT in D. melanogaster. Northern blot analysis revealed no diurnal variation of aaNAT2 transcription, similar to the results obtained for aaNAT1a and aaNAT1b.